Deciphering two rounds of cell lineage segregations during bovine preimplantation development
Deciphering two rounds of cell lineage segregations during bovine preimplantation development
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DOI：
10.1096/fj.202002762rr
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发表时间：
2021-10-01
期刊：
影响因子：
4.8
通讯作者：
Kawahara, Manabu
中科院分区：
文献类型：
--
作者：
Akizawa, Hiroki;Saito, Shun;Kawahara, Manabu
胚泡形成产生内细胞团（ICM）和滋养外胚层（TE），随后是ICM内的上胚层（Epi）和原始内胚层（PrE）的分化。虽然这些两轮细胞谱系分化在每种哺乳动物中都是正确的胚胎发生的基础，但它们的时空动态在物种之间是相当不同的。在这里，牛的囊胚阶段的分子细节解剖使用优化的体外培养方法。将胚泡胚胎置于充满营养丰富培养基的琼脂糖凝胶上，使胚胎暴露于气相和液相。在受精后第10天（D），将来自该“凝胶上”培养物的胚胎转移至代孕母亲，并成功植入。使用凝胶上培养的胚胎进行的免疫荧光研究显示，表达多能ICM标志物OCT 4的TE细胞的比例在D8时超过80%，在D9后迅速降低，并在D9.5时达到0%。第一个谱系分离过程与第二个谱系分离过程在时间上是平行的，通过表达SOX 2的Epi细胞和表达SOX 17的PrE细胞的空间分离来鉴定。来自D8体外受精胚胎和D14体内胚胎的TE细胞的RNA-seq比较显示，除了多能性相关基因的急剧减少外，TE细胞高度表达Wnt、FGF和VEGF信号通路相关基因，以促进胎儿-母体相互作用所需的功能成熟。凝胶上培养的TE细胞的定量PCR分析进一步证实了关键TE标志物表达的时间依赖性增量。总之，本研究提供了平台，了解哺乳动物植入前发育的物种特异性策略。
Blastocyst formation gives rise to the inner cell mass (ICM) and trophectoderm (TE) and is followed by the differentiation of the epiblast (Epi) and primitive endoderm (PrE) within the ICM. Although these two-round cell lineage differentiations underpin proper embryogenesis in every mammal, their spatiotemporal dynamics are quite diverse among species. Here, molecular details of the blastocyst stage in cattle were dissected using an optimized in vitro culture method. Blastocyst embryos were placed on agarose gel filled with nutrient-rich media to expose embryos to both gaseous and liquid phases. Embryos derived from this "on-gel" culture were transferred to surrogate mothers on day (D) 10 after fertilization and successfully implanted. Immunofluorescent studies using on-gel-cultured embryos revealed that the proportion of TE cells expressing the pluripotent ICM marker, OCT4, which was beyond 80% on D8, was rapidly reduced after D9 and reached 0% on D9.5. This first lineage segregation process was temporally parallel with the second one, identified by the spatial separation of Epi cells expressing SOX2 and PrE cells expressing SOX17. RNA-seq comparison of TE cells from D8 in vitro fertilized embryos and D14 in vivo embryos revealed that besides drastic reduction of pluripotency-related genes, TE cells highly expressed Wnt, FGF, and VEGF signaling pathways-related genes to facilitate the functional maturation required for feto-maternal interaction. Quantitative PCR analysis of TE cells derived from on-gel culture further confirmed time-dependent increments in the expression of key TE markers. Altogether, the present study provides platforms to understand species-specific strategies for mammalian preimplantation development.